(Note: Literature references on the following background information and on conventional test method and laboratory procedures well known to the ordinary person skilled in the art, and other such state-of-the-art techniques as used herein, are indicated in parentheses, and appended at the end of the specification.)
Secreted metalloproteases (MMPs) initiate tissue remodeling by degradation of extracellular matrix (ECM) macromolecules (reviewed in 1-3). Normal physiological processes such as morphogenesis, tissue repair, and angiogenesis, are dependent upon spatial and temporal regulation of the activity of these enzymes, while malignant cells exploit these same proteases to promote invasion and metastasis (4-7). A clear understanding of the mechanisms governing regulation of MMP activity in extracellular space has remained an elusive goal. The MT1-MMP/GelA system (8-16) provides a first glimpse at a mechanism by which an activity of a soluble MMP, GelA (17), can be spatially regulated via its recruitment to the cell surface where the GelA proenzyme is converted into its active form. Transfection of Cos1 cells with MT1-MMP is sufficient to cause GelA binding to the cell surface and its activation (8,19). The cell surface activation of GelA involves a two step proteolytic processing of its propeptide. The first cleavage of the Asn.sup.37 -Leu peptide bond is dependant on MT1-MMP (9), a membrane bound metalloprotease. This cleavage is also dependent on GelA having an intact C-terminal domain since a truncated form of the GelA proenzyme lacking a C-terminal domain can not be activated by membrane bound MT1-MMP (13). Consequently the exogenously added recombinant GelA-CTD is a competitive inhibitor of Asn.sup.37 -Leu cleavage (9,10). Finally this reaction is inhibited in the presence of an excess of inhibitor, TIMP-2, while TIMP-1 has no effect. The consequent cleavage of propeptide is accomplished via an autoproteolytic, MT1-MMP independent mechanism (9,10,18,19) to generate a 62 kDa active GelA with an amino-terminal residue Tyr.sup.81. These data demonstrate that binding of GelA to the cell surface via its CTD is a prerequisite for enzyme activation. We have previously shown that two closely related proenzymes GelA and B form specific complexes with TIMP-2 and TIMP-1 respectively (20). These complexes are also formed via inhibitor interaction with the carboxyl-end domain of proenzyme (21,22). Thus TIMP-2 and cell surface binding activities of GelA-CTD appear to be interrelated. We have purified activated form of MT1-MMP using affinity chromatography approach (9) and demonstrated that it acts as cell surface TIMP-2 receptor with Kd=1.65.times.10.sup.-9 M. The MT1-MMP-TIMP-2 complex in turn acts as a receptor for GelA-CTD (Kd=0.42.times.10.sup.-9 M). The data we have presented support the hypothesis that the cell surface binding of GelA-CTD occurs via formation of a tri-molecular complex of activated MT1-MMP/TIMP-2/pro-GelA that promotes pro-GelA activation. This model, however, does not satisfactory resolve the GelA activation mechanism for the following reasons. The inhibitor TIMP-2 consists of two domains. The amino-terminal, inhibitory domain interacts with the active center of MMPs to form an inhibitory complex (23,24). The C-terminal domain binds to GelA-CTD. Thus the inhibitory complex of TIMP-2 with activated MT1-MMP can leave the C-terminal domain of the inhibitor exposed and available for interaction with GelA-CTD. In fact we have reported an analogous tri-molecular complex between GelB, TIMP-1 and activated interstitial collagenase (22) where the collagenase component of the complex was inhibited. Moreover the specific inhibition of soluble form of MT1-MMP by TIMP-2 has been recently demonstrated (25,26). Thus, the model of cell surface GelA activation that requires assembly of the MT1-MMP/TIMP-2/pro-GelA complex leaves unanswered the question of how the MT1-MMP inhibited by TIMP-2 is able to cleave the Asn.sup.37 -Leu peptide bond to initiate activation of the pro-enzyme. An answer to this question demands a better understanding of the mechanism by which GelA-CTD interacts with TIMP-2 and MT1-MMP on the cell surface. We have recently reported the high resolution crystal structure of Gel A-CTD (27). Here we report the results of extensive alanine scanning mutagenesis of solvent exposed GelA-CTD amino-acid residues and, using the coordinates of the GelA-CTD structure, define a TIMP-2 binding site on the surface of this domain. By comparison of the TIMP-2 binding site to the same regions in related MMP structures, we characterize structural features required for general TIMP binding and the specificity of TIMP-2-GelA-CTD interaction. We also report analysis of GelA activation inhibition activity of GelA-CTD mutants relative to that of wild type.